Bi-specific antibodies for pre-targeting diagnosis and therapy

Abstract

The present invention relates to a bi-specific antibody or antibody fragment having at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable conjugate. The targetable conjugate comprises a carrier portion which comprises or bears at least one epitope recognizable by at least one arm of said bi-specific antibody or antibody fragment. The targetable conjugate further comprises one or more therapeutic or diagnostic agents or enzymes. The invention provides constructs and methods for producing the bi-specific antibodies or antibody fragments, as well as methods for using them.

1. A method of preparing a bi-specific Fab-scFv fusion protein having at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable conjugate which comprises a carrier portion which comprises or bears at least one epitope recognizable by said at least one other arm of said bi-specific antibody or antibody fragment, and one or more conjugated therapeutic or diagnostic agents, or enzymes, comprising:
 (1) (A) introducing into a mammalian host cell a recombinant DNA construct comprising an expression cassette capable of producing in said host cell a fragment of said bi-specific fusion protein, wherein said construct comprises, in the 5′ to 3′ direction of transcription, a transcriptional initiation regulatory region functional in said mammalian host cell, a translational initiation regulatory region functional in said mammalian host cell, a DNA sequence encoding a scFv linked to a Fd fragment, and a transcriptional and translational ter nation regulatory region functional in said mammalian host cell, wherein expression of said fragment of said bi-specific fusion protein is under the control of said regulatory regions;
 (B) co-introducing into said mammalian host cell a recombinant DNA construct comprising an expression cassette capable of producing in said mammalian host cell a light-chain antibody fragment which is complementary to said Fd fragment in (A) and which when associated with said Fd fragment forms a Fab fragment whose binding site is specific for said targeted tissue, wherein said construct comprises, in the 5′ to 3′ direction of transcription, a transcriptional initiation regulatory region functional in said mammalian host cell, a translational initiation regulatory region functional in said mammalian host cell, a DNA sequence encoding a light-chain antibody fragment, and a transcriptional and translational termination regulatory region functional in said mammalian host cell, wherein expression of said light-chain antibody fragment is under the control of said regulatory regions; 
 (C) growing said cell; and 
 (D) isolating said bi-specific Fab-scFv fusion protein, or 
 
 (2) (A) introducing into a first mammalian host cell a recombinant DNA construct comprising an expression cassette capable of producing in said first mammalian host cell a fragment of said bi-specific fusion protein, wherein said construct comprises, in the 5′ to 3′ direction of transcription, a transcriptional initiation regulatory region functional in said first mammalian host cell, a translational initiation regulatory region functional in said first mammalian host cell, a DNA sequence encoding a scFv linked to a Fd fragment, and a transcriptional and translational termination regulatory region functional in said first mammalian host cell, wherein expression of said fragment of said bi-specific fusion protein is under the control of said regulatory regions; 
 (B) introducing into a second mammalian host cell a recombinant DNA construct comprising an expression cassette capable of producing in said second mammalian host cell a light-chain antibody fragment which is complementary to said Fd fragment in (2)(A) and which when associated with said Fd fragment forms a Fab fragment whose binding site is specific for said targeted tissue, wherein said construct comprises, in the 5′ to 3′ direction of transcription, a transcriptional initiation regulatory region functional in said second mammalian host cell, a translational initiation regulatory region functional in said second host cell, a DNA sequence encoding a light-chain antibody fragment, and a transcriptional and translational termination regulatory region functional in said second mammalian host cell, wherein expression of said light-chain antibody fragment is under the control of said regulatory regions;
 (C) growing said first and second mammalian host cells; 
 (D) optionally isolating said bi-specific fusion protein fragment and said light-chain antibody fragment; 
 (E) combining said fragments to produce a Fab-scFv bi-specific fusion protein; and 
 (F) isolating said bi-specific fusion protein. 
 
 
     
     
       2. The method of  claim 1 , wherein said at least one arm that specifically binds a targeted tissue is a humanized Fab fragment. 
     
     
       3. The method of  claim 1 , wherein said at least one other arm specifically binds said epitope of said targetable conjugate, and said epitope comprises a peptide. 
     
     
       4. The method of  claim 1 , wherein said at least one other arm specifically binds said epitope of said targetable conjugate, and said epitope comprises a carbohydrate. 
     
     
       5. The method of  claim 1 , wherein said at least one other arm specifically binds said epitope of said targetable conjugate, and said epitope comprises a hapten. 
     
     
       6. The method of  claim 1 , wherein said at least one other arm specifically binds said epitope of said targetable conjugate, and said epitope comprises a chelator or a metal-chelate complex. 
     
     
       7. The method of  claim 6 , wherein said chelator is a hard base chelator for a hard acid cation. 
     
     
       8. The method of  claim 6 , wherein said chelator is a soft base chelator for a soft acid cation. 
     
     
       9. The method of  claim 7 , wherein said chelator is a hard base chelator that comprises carboxylate and amine groups. 
     
     
       10. The method of  claim 7 , wherein said hard base chelator is DTPA, NOTA, DOTA or TETA. 
     
     
       11. The method of  claim 1 , wherein said at least one other arm specifically binds a tyrosyl-lysine dipeptide. 
     
     
       12. The method of  claim 1 , wherein said at least one other arm specifically binds Tyr-Lys(DTPA)-N11 2 , or Lys(DTPA)-Tyr-Lys(DTPA)-NH 2 . 
     
     
       13. A method of preparing a bi-specific Fab-scFv fusion protein having at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targetable conjugate which comprises a cater portion which comprises or bears at least one epitope recognizable by said at least one other arm of said bi-specific antibody or antibody fragment, and one or more conjugated therapeutic or diagnostic agents, or enzymes, comprising:
 (1) (A) introducing into a mammalian host cell a recombinant DNA construct comprising an expression cassette capable of producing in said mammalian host cell a fragment of said bi-specific fusion protein, wherein said construct comprises, in the 5′ to 3′ direction of transcription, a transcriptional initiation regulatory region functional in said mammalian host cell, a translational initiation regulatory region functional in said mammalian host cell a DNA sequence encoding a scFv linked to a light-chain antibody fragment, and a transcriptional and translational termination regulatory region functional in said mammalian host, cell, wherein expression of said fragment of said bi-specific fusion protein is under the control of said regulatory regions; 
 (B) co-introducing into said mammalian host cell a recombinant DNA construct comprising an expression cassette capable of producing in said mammalian host cell a Fd fragment which is complementary to said light-chain antibody fragment in (A) and which when associated with said light-chain antibody fragment forms a Fab fragment whose binding site is specific for said targeted tissue, wherein said construct comprises, in the 5′ to 3′ direction of transcription, a transcriptional initiation regulatory region functional in said mammalian host cell, a translational initiation regulatory region functional in said host cell, a DNA sequence encoding a Fd fragment, and a transcriptional and translational termination regulatory region functional in said mammalian host cell, wherein said expression of Fd fragment is under the control of said regulatory regions; 
 (C) growing said cell; and 
 (D) isolating said bi-specific Fab-scFv fusion protein, or 
 (2) (A) introducing into a first mammalian host cell a recombinant DNA construct comprising an expression cassette capable of producing in said first mammalian host cell a fragment of said bi-specific fusion protein, wherein said construct comprises, in the 5′ to 3′ direction of transcription, a transcriptional initiation regulatory region functional in said first mammalian host cell, a translational initiation regulatory region functional in said first mammalian host cell, a DNA sequence encoding a scFv linked to a light-chain antibody fragment, and a transcriptional and translational termination regulatory region functional in said first mammalian host cell, wherein expression of said fragment of said bi-specific fusion protein is under the control of said regulatory regions; 
 (B) introducing into a second mammalian host cell a recombinant DNA construct comprising an expression cassette capable of producing in said second mammalian host cell a Fd fragment which is complementary to said light-chain antibody fragment in (2)(A) and which when associated with said light-chain antibody fragment forms a Fab fragment whose binding site is specific for said targeted tissue, wherein said construct comprises, in the 5′ to 3′ direction of transcription, a transcriptional initiation regulatory region functional in said second mammalian host cell, a translational initiation regulatory region functional in said second mammalian host cell, a DNA sequence encoding a Fd fragment, and a transcriptional and translational termination regulatory region functional in said second mammalian host cell, wherein expression of said Fd fragment is under the control of said regulatory regions; 
 (C) growing said first and second mammalian host cells; 
 (D) optionally isolating said bi-specific fusion protein fragment and said Fd fragment; and 
 (E) combining said fragments to produce a bi-specific Fab-scFv fusion protein; and 
 (F) isolating said bi-specific fusion protein.